Functional processing of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N): evidence for a critical role of proteolytic processing in the regulation of its catalytic activity, subcellular localization and substrate targeting in vivo.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear homolog CaMKP-N are Ser/Thr protein phosphatases that belong to the PPM family. These phosphatases are highly specific for multifunctional CaM kinases and negatively regulate their activities. CaMKP-N is only expressed in the brain and specifically localized in the nucleus.

A minimum size homologue of Ca2+/calmodulin-dependent protein kinase IV naturally occurring in zebrafish.

Ca(2+)/calmodulin-dependent protein kinase (CaMK) IV is a multifunctional Ser/Thr protein kinase that is predominantly expressed in the nuclei of neurons. CaMKIV consists of a catalytic domain and a regulatory (Ca(2+)/calmodulin binding and autoinhibitory) domain, which are located in the N-terminal and central regions, respectively. Here, we identified the zebrafish homologue of CaMKIV (zCaMKIV) on the basis of biochemical characterization.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is indispensable for normal embryogenesis in zebrafish, Danio rerio.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). However, the biological functions of this enzyme have not been clarified in vivo. To investigate the biological significance of CaMKP during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP (zCaMKP).

Inhibitors of the Ca(2+)/calmodulin-dependent protein kinase phosphatase family (CaMKP and CaMKP-N).

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear isoform CaMKP-N are unique Ser/Thr protein phosphatases that negatively regulate the Ca(2+)/calmodulin-dependent protein kinase (CaMK) cascade by dephosphorylating multifunctional CaMKI, II, and IV. However, the lack of specific inhibitors of these phosphatases has hampered studies on these enzymes in vivo. In an attempt to obtain specific inhibitors, we searched inhibitory compounds and found that Evans Blue and Chicago Sky Blue 6B served as effective inhibitors for CaMKP.

Expression, characterization, and gene knockdown of zebrafish doublecortin-like protein kinase.

Doublecortin-like protein kinase (DCLK) is a protein Ser/Thr kinase expressed in brain and believed to play crucial roles in neuronal development. To investigate the biological significance of DCLK, we isolated cDNA clones for zebrafish DCLK (zDCLK) and found that there were five splice variants of the kinase. In this study, the catalytic properties of a major isoform of zDCLK, which we designated as zDCLK1, and of an N-terminal truncated mutant retaining the kinase domain were examined by expressing them in Escherichia coli.

Knockdown of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish.

Nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N) is an enzyme that dephosphorylates and concomitantly downregulates multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) in vitro. However, the functional roles of this enzyme in vivo are not well understood. To investigate the biological significance of CaMKP-N during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP-N (zCaMKP-N).

Mutational analysis of Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP).

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a member of the serine/threonine protein phosphatases and shares 29% sequence identity with protein phosphatase 2Calpha (PP2Calpha) in its catalytic domain. To investigate the functional domains of CaMKP, mutational analysis was carried out using various recombinant CaMKPs expressed in Escherichia coli. Analysis of N-terminal deletion mutants showed that the N-terminal region of CaMKP played important roles in the formation of the catalytically active structure of the enzyme, and a critical role in polycation stimulation.

Identification of major Ca(2+)/calmodulin-dependent protein kinase phosphatase-binding proteins in brain: biochemical analysis of the interaction.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a unique protein phosphatase that specifically dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). To clarify the physiological significance of CaMKP, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose bisphosphate aldolase as major binding partners of CaMKP in a soluble fraction of rat brain using the two-dimensional far-Western blotting technique, in conjunction with peptide mass fingerprinting analysis. We analyzed the affinities of these interactions.